Recombinant adeno-associated virus (rAAV) has several characteristics that underscore its potential as a gene therapy vector for numerous target organs and inherited or acquired diseases, a vaccine vector or for diagnostics. Moreover, rAAV vector systems potentially offer major advantages over other gene delivery vehicles, including adenoviruses and retroviruses. These include the ability of rAAV to readily transduce non dividing or slowly dividing cells and persist essentially for the lifetime of the cell, the lack of undesirable cellular immune responses since all viral genes can be deleted from the vector, and the fact that AAV has never been associated with human disease.
A serotype 2 rAAV (rAAV-2) vector expressing the CFTR gene was the first AAV vector to be utilized in clinical trials. This vector has demonstrated promise in patients with cystic fibrosis and has advanced to phase II trials (Flotte et al., 1996; Wagner et al., 1999; Wagner et al., 2002; Aitken et al., 2001). In recent years, additional rAAV 2 vectors have been or are currently being advanced to clinical trials to treat a number of disease states including a rAAV2 factor IX vector in phase I clinical trials in patients with hemophilia B (Kay et al., 2000), and a rAAV2 sarcoglycan vector in phase I clinical trials of patients with CNS disease. Additionally, clinical trials for rAAV expressing proteins to treat Parkinsons disease (RDAC, 2001), and Canavan's disease (Janson et al., 2001), have been proposed.
Other serotypes of AAV are known to exist, although they are all closely related at the functional, structural, and genetic level (see, e.g., Blacklow, 1988; and Rose, 1974). All AAV serotypes exhibit very similar replication properties mediated by homologous rep genes; all bear three related capsid proteins such as those expressed in AAV-2, and all contain 5′-3′ ITR sequences. Currently, 8 serotypes of AAV have been described with the complete genome sequence information available for AAV-1-AAV-6 (Srivastava et al., 1983; Miramatsu et al., 1996; Chiorini et al., 1967; Xiao et al., 1999; Chiorini et al., 1999; Bantel-Schaal et al., 1999; Rutledge et al., 1998) and capsid gene sequence for AAV-7 and AAV-8 (Gao et al., 2002). AAV-6 has been shown to be a recombinant between AAV-1 and AAV-2. In addition, there are two isolates and sequences of AAV-3 that differ from each other in a number of amino acids in both rep and cap (Rutledge et al., 1998). AAV-5 is the most distantly related of the serotypes, and displays a serotype-specific terminal resolution site (trs) in its ITR (Chiorini et al., 1999). Even though rep proteins from other serotypes bind the AAV-5 ITR, they do not efficiently cleave at the trs. In addition to recent developments in AAV and rAAV serotypes, numerous groups are experimenting with rAAV pseudotypes.
Variations in cell surface receptor usage for binding of rAAV to cell membranes exists among various serotypes and may be in part responsible for the differences in transduction efficiencies in various tissue and cell types. Although conflicting data exists, it has become apparent that there are differences among the serotypes in the efficiency of transgene expression in various tissues and cell types. For example, rAAV-1 and rAAV-7 overall appear several orders of magnitude superior for transduction of murine muscle tissue although rAAV-5 also demonstrates enhancement compared to rAAV-2 (Gao et al., 2002; Chao et al., 2000; Rabinowitz et al., 2002; Hildinger et al., 2001). rAAV-8 transduces murine liver up to 100-fold higher (Gao et al., 2002) than rAAV-2, and rAAV-5 appears superior in transduction of cells of the murine respiratory tract (Zabner et al., 2000; Aurichio et al., 2002). rAAV-5 generally appears to be superior to rAAV-2-based vectors in all tissue types tested so far including CNS, muscle, liver and retina (Chao et al., 2000; Rabinowitz et al., 2002; Hildinger et al., 2001; Aurichio et al., 2002; Davidson et al., 2000; Mingozzi et al., 2002). Similarly, rAAV-6 is more efficient than rAAV-2 in transducing murine airway epithelia and alveoli, while rAAV-3 is superior in transducing smooth muscle cells (Halbert et al., 2001; Halbert et al., 2000). rAAV-4 transduces ependymal cells in the murine CNS almost exclusively, while rAAV-5 transduces both neurons and astrocytes (Davidson et al., 2000). In retina, a number of studies have demonstrated large differences among serotypes in the ability to transduce photoreceptor cells and the retinal pigmented epithelium (Walters et al., 2001; Aurricchio et al., 2001; Yang et al., 2002).
Despite the fact that rAAV has a very broad host tropism in a variety of human, simian, and rodent cell lines (Lebkowski et al., 1988; Muzyczka, 1992), the overall transduction efficiency in human airway epithelia and other tissues seems to be quite low. Previous studies have suggested that single to double strand conversion of the rAAV genome may be the rate-limiting step for AAV-mediated gene transfer (Ferrari et al., 1996; Fisher et al., 1996). These studies demonstrated that adenovirus E4orf6 enhances the conversion of single-stranded DNA genomes to linear, double-stranded replication form dimers (Rfd) and monomers (Rfm), through a pathway characteristic of the lytic phase of rAAV replication. The structure of these replication forms consists of head-to-head and tail-to-tail orientated linear concatamers with one covalently linked end (Ferrari et al., 1996; Fisher et al., 1996). In contrast, recent studies have elucidated an alternative pathway for the conversion of rAAV genomes to double-stranded circular intermediates with head-to-tail monomer and concatamer structures (Duan et al., 1999; Duan et al., 1998; Sanlioglu et al., 1999). The distinct pathways leading to the formation of either circular AAV genomes or Rf intermediates appear to be regulated by different cellular factors. For example, adenoviral E4orf6 expression decreases circular genome formation while adenovirus E2a enhances its formation (Duan et al., 1999). Similarly, UV irradiation also enhances AAV circular intermediate formation but not Rf intermediates (Sanlioglu et al., 1999).
More recently, when cellular binding protein FK506-(FKBP-52) was phosphorylated at tyrosine residues (by the epidermal growth factor receptor protein tyrosine kinase), FKBP-52 was demonstrated to be bound to the single-stranded D-sequence of the AAV ITR causing an impairment in second strand synthesis (Qing et al., 2001; Qing et al., 2003. The efficiency of rAAV transduction in a number of cell types in vitro and in vivo correlates with the phosphorylation state of FKBP-52. For example, in HeLa cells, overexpression of a cellular phosphatase (TC-PTP), led to dephosphorylation of the FKBP-52, an increase in AAV second-strand DNA synthesis, and an increase in transgene expression. Transgenic mice expressing either the wild type (wt) or a catalytically mutant form of TC-PTP, were created. Hematopoietic stem cells from transgenic mice expressing the wt TC-PTP phosphatase were transduced by a rAAV2, but those from the phosphatase-negative mutant were not. These results suggest that the block to second-strand DNA synthesis is due to binding of FKBP-52 to the D-sequence of infecting vector genomic DNA and that this binding is regulated by phosphorylation. Thus, numerous strategies aimed at increasing the transduction frequency for AAV have focused on enhancing the molecular conversion of nonfunctional viral genomes to expressible forms (Fisher et al., 1996; Sanlioglu et al., 1999) or by increasing transcription and translation efficiencies by altering the transgene expression cassettes (Zabner et al., 1996; Xiao et al., 1998).
A second approach aimed at improving transduction efficiencies of rAAV has focused on the binding of rAAV to cell surface receptors. Many primary and secondary cell surface receptor molecules have been identified for the various AAV serotypes. The primary receptors identified (heparin sulfate and sialic acid) are found on many cell types and are also utilized by a large number of viruses besides AAV. This suggests that additional receptors that lend more specificity to attachment and penetration of cells might exist and several such co-receptors have been identified. Thus, additional strategies to improve rAAV transduction efficiency have focused on manipulation of cell surface receptors (Qing et al., 1997) and/or receptor ligands in the virus coat proteins (Wickham et al., 1996a; Wickham et al., 1996b; Bartlett et al., 1999).
While binding to the cell surface membrane and successful conversion to a double stranded DNA genome are important, the efficiency of these events does not necessarily correlate with the overall ability or efficiency of rAAV to transduce a given cell type. This has been increasingly apparent in recent years as a more detailed understanding of the trafficking and uncoating of rAAV has been accumulated (Duan et al., 1998; Seisenberger et al., 2001; Hanson et al., 2001; Bantel-Schaal et al., 2002; Yan et al., 2002). For example, polarized human airway epithelial cells are transduced with varying efficiencies by rAAV-2 depending on the route of delivery; entry from the basolateral surface results in about a 200-fold increase in gene expression in the cells compared to vector administered from the apical surface (Duan et al., 1998; Duan et al., 2000). Surprisingly, the difference in rAAV cell surface binding between two cell surfaces is only about 5-fold. This finding led to the discovery that the vectors traffic differently in these cells depending on the route of delivery (Duan et al., 1998; Duan et al., 2000).
Previous reports have clearly demonstrated that intracellular trafficking to the nucleus for rAAV-2 and canine parvovirus is a slow, rate-limiting process for certain cell types (Parker et al., 2000; Hanson et al., 2001; Hanson et al., 2000; Duan et al., 2000). Canine parvovirus and rAAV-2 have also been demonstrated to be endocytosed through clathrin-dependent receptor endocytosis and processed through endosomal compartments in a similar fashion to transferrin, but not a fluid phase marker such as dextran (Parker et al., 2000; Bartlett et al., 2000; Benson et al., 2000; Duan et al., 1999). Transferrin trafficking has been extensively studied and shown to move through the early endosome to perinuclear recycling endosome (PNRE) (Sonnichsen et al., 2000; Ren et al., 1998). The recycling of transferrin through the PNRE requires the coordinated interactions of several small GTPases (Rab5, Rab4, and Rab11) which direct the movement and fusion of early endosomes to the PNRE compartment (Sonnichsen et al., 2000).
Studies designed to develop agents to improve the efficiency of rAAV transduction have demonstrated that proteosome inhibitors such as the tripeptides LLnL and Z-LLL can enhance transduction of rAAV. Agents of this class affect ubiquitination of rAAV by inhibiting calpains, cathepsins, cysteine proteases as well as the chymotrypsin-like protease activity of proteasomes in polarized cell types (Duan et al., 2000; Yan et al., 2002). Additionally, agents affecting DNA metabolism including hydroxyurea, novobiocin, amsacrine, and etopside were tested for the ability to enhance rAAV transduction based on the hypothesis that these drugs would increase the rate of conversion of the single stranded rAAV genome to a double stranded form. Results demonstrated that etoposide, hydroxyurea, and campothecin were effective at enhancing rAAV transduction when utilized singularly but when used in combination produced no additive or synergistic effects (Russel et al., 1995). Furthermore, these agents were only stated as effective in enhancing rAAV transduction in cell types for which gene conversion is rate limiting. Steps which proceed gene conversion (i.e., intracellular trafficking and processing of virions) appear to be critical rate-limiting steps in primary cells and differentiated tissues such as the airway (Hansen et al., 2000; Duan et al., 2000).
There exists a need for improved transduction efficiencies for rAAV vectors. Thus, what is needed is the identification of agents that can alter, e.g., increase or enhance, rAAV transduction or rAAV transduction frequencies in vivo. What is also needed is the identification of agents that increase or enhance the expression of a rAAV heterologous transgene in non-dividing or slowly dividing cells or tissues, such as those in the liver and the airway.